Small RNA Research

microRNA and Diseases

MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression by translational inhibition and destabilization of mRNAs [1]. Thousands of miRNAs have been currently identified in human, but only a small fraction of them have validated functional roles. Among them, the miRNAs that are involved in human diseases, especially in cancers, have attracted a great deal of interest.

MicroRNAs in Cancer

The aberrant expression of microRNAs in human tumors is not just a casual association, but they can exert a causal role, as oncogenes or tumor suppressors, in different steps of the tumorigenic process, from initiation and development to progression toward the acquisition of a metastatic phenotype [2].

Figure 1. Schematic representation of microRNAs that are involved in cancer initiation and progression [3].

Tumor Initiation and Growth

Deregulation of cell proliferation, differentiation, and apoptosis represents one of the main hallmarks of cancer initiation and progression. miRs take part in this process by controlling different steps and affecting integrated pathways involved in carcinogenesis. miR-15 and miR-16 represent clear examples being located at a crossway between apoptosis and cell cycle control. miR-15a and miR–16–1, whose loci are deleted in more than half of cases of B-CLL and in advanced prostate cancers, physiologically control the expression of the anti-apoptotic gene B-cell lymphoma 2 (BCL2) through its post-transcriptional inhibition. miR-15a and miR–16–1 loss explains BCL2 overexpression in both B–CLL and prostate cancers.

Additional evidence highlights that miR–15a and miR–16–1 might control other genes involved in the programmed cell death pathway and cell cycle checkpoints such as CCND1 (encoding cyclin D1) and WNT3A, promoting several tumorigenic features, including survival, proliferation, and invasion.

In physiologic conditions, miR expression is finely tuned and often involved in feedback loops that control cell cycle homeostasis; the miR-34 family represents a paradigmatic example. MiR-34a was first described as a potential tumor-suppressor in neuroblastoma. Low levels of miR-34a expression were shown in pancreatic cancer cells, colon, ovarian, and lung cancers. miR-34a and miR-34b/c expression can be induced by DNA damage and oncogenic stress in a p53-dependent manner. The introduction of either miR-34a or miR- 34b/c in normal human fibroblasts leads to substantial inhibition of cell growth, which might be partially due to miR-34a– dependent MYCN inhibition. On the other end, miR-34a indirectly controls p53 activation through SIRT1. Inhibition of SIRT1 by miR-34 leads to an increase in acetylation of p53 and expression of p21 and PUMA.

let-7 miRs are highly conserved in invertebrates and vertebrates, and many members of let-7 family map to regions altered or deleted in human tumors, indicating that these genes may function as tumor-suppressors. The let-7 family has been reported to be downregulated in human lung cancer, colon cancer, and lymphoma. let-7 has been demonstrated to act as a tumor-suppressor by directly silencing cell cycle proto-oncogenes, such as RAS and HMGA2. Other direct let-7 targets include genes implicated in cell-cycle regulation, including CDC25a, CDK6, and CCND. Recent evidences have demonstrated that let-7a acts as a tumor-suppressor in prostate cancer by downregulating E2F2 and cyclin D2. [3]

Angiogenesis

During tumor progression, the "angiogenic switch" is a key step for the expansion of a tumor mass. This switch is primarily activated when a growing tumor mass surpasses the maximal volume that can be sustained by diffusion of oxygen and nutrients. Low oxygen tension in large burden tumors induces cancer cells to overexpress the hypoxia-inducible transcription factor 1 (HIF-1). This transcription factor binds to hypoxia-response elements (HREs) located upstream of target genes, and activates numerous hypoxia-response genes, such as the pro-angiogenic growth factor, vascular endothelial growth factor (VEGF).

miRs affecting angiogenesis, also called "angiomiRs", have been shown to control and promote angiogenesis. Among these, miR-126, which is highly expressed in endothelial cells, was shown to regulate many aspects of endothelial cell biology, including cell migration, reorganization of the cytoskeleton, capillary network stability, and cell survival. In detail, miR-126 operates by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1 and the phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-beta).

The MYC-activated miR–17–92 cluster was among the first miRs linked to tumor angiogenesis. The miR–17–92 cluster is a typical example of a polycistronic miR cluster encoding for miR-17, miR-18a, miR-19a/b, miR-20a, and miR- 92a, which are highly expressed in several tumors. Overexpression of the entire miR–17–92 cluster in myc-induced tumors increases angiogenesis by a paracrine mechanism. This pro-angiogenic function has been attributed to the downregulation of the anti-angiogenic TSP-1 and of the connective tissue growth factor (CTGF), targeted by miR-18 and miR-19.

Moreover, hypoxic reduction of miR-16, miR-15b, miR-20a and miR-20b, which directly repress Vegf, accounts for the sustained expression of Vegf during hypoxia and supports the angiogenic process.

Figure 2. Schematic representation of miRNAs that are involved in angiogenesis [4].

Metastasis

The metastatic process includes complex and multiple steps: cell motility, tissue invasion, intravasation, translocation through the blood and lymph system, extravasation, and initial microscopic proliferation at a new site. Cancer-associated miRs harbor anti- or pro-metastatic properties by multiple signalling pathways and targeting various proteins that are major players in this process. One of the most important and controversial miRs involved in metastasis control is miR-10b, which is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion. miR-10b is transcriptionally activated by the pro-metastatic transcription factor Twist1 and is essential for Twist1-induced epithelial–mesenchymal transition. HoxD10 has been identified as another miR- 10b target and its downregulation results in the expression of pro-metastatic products, including RHOC which is involved in cell migration and extracellular matrix remodeling. miR-21 seems to be involved in several steps of the metastatic process. The biological effects of mir-21 are probably due to the simultaneous repression of multiple tumor-suppressor genes, including Tropomisoin (TPM)1, programmed cell death protein (PDCD) 4, maspin, and PTEN. miR-21 also regulates metalloproteinases (MMPa) by directly controlling the MMP inhibitor RECK, whose expression is prognostic in a number of common cancers.

The early stages of metastasis are characterized by loss of both cell– cell and cell–matrix contact and switch from a collective invasion pattern to a detached and disseminated cell migration method. This process is called epithelial-mesenchymal transition (EMT), in which epithelial cells acquire a fibroblast-like morphology. The miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) and miR-205 are downregulated in cells that have undergone EMT in response to transforming growth factor (TGF)-beta or to ectopic expression of the protein tyrosine phosphatase Pez. Loss of miR-205 and miR-200 family members in tumours can be mainly ascribed to the repressor activity of the ZEB family of transcription factors, which regulate EMT-related genes such as epithelial (E)-cadherin, mucin, tight junction protein ZO3, connexin 26 and plakophilin 2. microRNAs involved in stemness maintenance also contribute to a metastatic phenotype. A well characterized example of the potential dual role of miRNAs in stemness and metastasis is represented by miR-101. Through direct inhibition of EZH2, an epigenetic regulator of the polycomb group proteins with important functions in embryonic stem cell regulation, miR-101 can control tumour cell proliferation, but also tumour cell invasiveness and metastatic ability.

A further example is the miR-221 and miR-222 cluster, which share the same seed sequence. Loss of miR-221 and miR-222 in endothelial cells sustains the proliferative and angiogenic properties of KIT, whereas their commonly observed upregulation in tumour cells, through suppression of the cyclin-dependent kinase inhibitor p27, increases their proliferative as well as their metastatic potential.

Besides cancers, microRNAs have also been implicated in many other human diseases, including cardiovascular diseases, neurodevelopmental diseases, autoimmune diseases, liver diseases and skeletal muscle diseases.

MicroRNAs in Cardiovascular Diseases (Figure 8 and 9)

The homoeostasis of the vascular system depends on the functionality of endothelial cells and coordinated regulation of angiogenesis, vasculogenesis, and vessel regression. The discovery of microRNAs in recent years has made it evident that these RNA molecules play important roles in regulation of heart function and have been linked to cardiovascular diseases, such as cardiac hypertrophy (Figure 8) and Ischemic heart disease (Figure 9).

Figure 8. A diagram showing miRNAs and their targets in cardiac hypertrophy [5].

Figure 9. A diagram showing miRNAs and their targets in cardiac ischemia [5].

MicroRNAs in neurodegenerative disease (Table 3)

Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease (PD), and other neurodegenerative disorders are a major health problem in both developed and developing countries. Accumulating evidences hve demonstrated that miRNAs are important contributors to neurodegenerative diseases (Table 3).[8]

To facilitate researchers' study, Arraystar has newly released the Human Cancer focus 384HC microRNA PCR Array, which contains 184 miRNAs involved in cancers, as well as some other major human diseases. By carefully collecting the critical miRNAs from multiple landmark publications, this array provides researchers with a convenient way to quickly analyze the miRNAs most relevant to cancers.